Substance P (SP) is a neuropeptide involved in physiological regulation and has also been implicated in the pathway resulting in pain and inflammation. In the previous funding period we showed that the SP neurokinin-1 receptor (NK-1R) desensitizes more rapidly and extensively than may other G protein-coupled receptor and that this is caused by receptor phosphorylation and is correlated with receptor-mediated physiological responses. In addition, we showed that the NK-1R undergoes vigorous agonist-dependent endocytosis and recycling as a primary mechanism of resensitization. We also showed that the NK-1R in the rat intestine mediates Clostridium difficile toxin A-induced inflammation, secretion, and tissue damage. These observations suggest that the NK-1R is highly regulated and plays a major role in intestinal inflammation, but the mechanisms of NK-1R regulation are incompletely understood. The current proposal describes studies designed to reveal additional mechanisms of NK-1R regulation. The proposed specific aims are to 1) test the hypothesis that the N-terminus of SP plays a role in homologous desensitization of the NK-1R even though the C-terminus contains all of the agonist activity of the peptide, 2) test the hypothesis that the cytoplasmic microfilament component of the cytoskeleton in NK-1R-expressing cells plays a role in SP-mediated signaling, and 3) test several hypotheses concerning the mechanisms of SP-stimulation of MAP Kinase activity via the NK-1R. These hypotheses will be tested by multiple approaches including assessment of NK-1R signaling and desensitization after site-directed NK-1R mutagenesis. These studies will lead to insight into the normal mechanisms of SP NK-1R regulation and will suggest possible mechanisms of abnormal NK-1R regulation in intestinal inflammatory diseases.